The present invention is directed to methods for oxidizing an H-phosphonate internucleoside linkage in a compound which has at least one 2xe2x80x2-substituent group. This invention further relates to methods for the preparation of mixed backbone oligomeric compounds having internucleoside linkages including phosphodiester, phosphorothioate, phosphoramidate and boranophosphate linkages. The present invention also relates to synthetic intermediates useful in such methods. Also included are mixed backbone oligomeric compounds having phosphodiester and boranophosphate internucleoside linkages or phosphodiester and boranophosphate internucleoside linkages in combination with other internucleoside linkages such as phosphorothioate and phosphoramidate internucleoside linkages. The methods of the present invention include a novel oxidation step that allows oxidation of a region having one or more H-phosphonate is internucleoside linkages to phosphodiester internucleoside linkages without degradation of adjacent phosphorothioate, phosphoramidate or boranophosphate internucleoside linkages.
Oligonucleotides and their analogs have been developed and used in molecular biology in a variety of procedures as probes, primers, linkers, adapters, and gene fragments. Modifications to oligonucleotides used in these procedures include labeling with nonisotopic labels, e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase or other reporter molecules. Other modifications have been made to the ribose phosphate backbone to increase the nuclease stability of the resulting analog. Examples of such modifications include incorporation of methyl phosphonate, phosphorothioate, or phosphorodithioate linkages, and 2xe2x80x2-O-methyl ribose sugar units. Further modifications include those made to modulate uptake and cellular distribution. With the success of these compounds for both diagnostic and therapeutic uses, there exists an ongoing demand for improved oligonucleotides, their analogs and synthetic processes for their preparation.
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. For disease states, classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect may be obtained with minimal side effects. It is therefore a general object of such therapeutic approaches to interfere with or otherwise modulate gene expression, which would lead to undesired protein formation.
One method for inhibiting specific gene expression is via the use of oligonucleotides, especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence. Several oligonucleotides are currently undergoing clinical trials for such use. Phosphorothioate oligonucleotides are presently being used as such antisense agents in human clinical trials for various disease states, including use as antiviral agents.
Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate their action. Several recent reports describe such interactions. Bielinska, et al., Science, 1990, 250, 997-1000; and Wu, et al., Gene, 1990, 89, 203-209.
In addition to their use as indirect and direct regulators of proteins, oligonucleotides and their analogs also have found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with inhibition of gene expression, diagnostic applications utilize the ability of oligonucleotides and their analogs to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence-specific hydrogen bonding of oligomeric compounds via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides and their analogs are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides and their analogs as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides and their analogs, both natural and synthetic, are employed as primers in such PCR technology.
Oligonucleotides and their analogs are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., Sambrook et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, Ausubel et al., Eds., Current Publications, 1993. Such uses include as synthetic oligonucleotide probes, in screening expression libraries with antibodies and oligomeric compounds, DNA sequencing, in vitro amplification of DNA by the polymerase chain reaction, and in site-directed mutagenesis of cloned DNA. See Book 2 of Molecular Cloning A Laboratory Manual, supra. See, also, xe2x80x9cDNA-protein interactions and The Polymerase Chain Reactionxe2x80x9d in Vol. 2 of Current Protocols In Molecular Biology, supra.
Oligonucleotides and their analogs can be synthesized to have customized properties that can be tailored for desired uses. Thus a number of chemical modifications have been introduced into oligomeric compounds to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include those designed to increase binding to a target strand (i.e., increase their melting temperatures, Tm), to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides and their analogs, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.
The chemical literature discloses numerous processes for coupling nucleosides through phosphorous-containing covalent linkages to produce oligonucleotides of defined sequence. One such method utilizes H-phosphonate monomers to prepare oliogmeric compounds. The standard H-phosphonate method has been used for the synthesis of uniformly modified oligomeric compounds containing phosphodiester, phosphorothioate or phosphoramidate internucleoside linkages. The method has not been effective for the synthesis of mixed backbone oligomers because during the oxidation step previously oxidized regions in an oligomer are degraded. Such degradation has been observed for the oxidation of oligomers having phosphorothioate and or phosphoramidate internucleoside linkages when further H-phosphonate internucleoside linkages are oxidized to phosphodiester linkages.
H-phosphonate methods and techniques are disclosed in numerous publications. See, Mackie and Hogrefe, Glen Research, H-phosphonate Chemistry, Glen Research Corp, Va.; Wada et al., J. Am. Chem. Soc., 1997, 119, 12710-12721; and Sergueev et al., J. Am. Chem. Soc., 1998, 120, 9417-9427.
Phosphoramidate oligodeoxynucleotides have been prepared using H-phosphonate chemistry. Dagle et al., Nucleic Acids Research, 1991, 19, 1805-1810.
2xe2x80x2-Methoxymethyl oligonucleotides have been prepared using H-phosphonate monomers. Bizdena et al., Collect. Czech. Chem. Commun., 1996, 61, 283-286.
The present invention provides methods for oxidizing H-phosphonate internucleoside linkages in a compound which bears at least one 2xe2x80x2-substituent group. This oxidation is effected by an oxidizing solution comprising an oxidizing agent, an aprotic solvent, a base and water.
The present invention also provides methods for the preparation of mixed backbone oligomeric compounds of formula: 
wherein:
each Z is, independently, a phosphodiester, phosphorothioate, phosphoramidate or boranophosphate internucleoside linkage;
each T1 and T2 is, independently, hydroxyl or a protected hydroxyl;
Bx is a heterocyclic base moiety;
each R1 is, independently, H, hydroxyl, a protected hydroxyl, a 2xe2x80x2-substituent group or a protected 2xe2x80x2-substituent group; and
n is an integer greater than 1;
provided that at least one of said Z is a phosphodiester internucleoside linkage and at least another of said Z is a phosphorothioate, phosphoramidate or boranophosphate internucleoside linkage;
comprising the steps of:
(a) providing a compound of formula: 
xe2x80x83wherein:
Pg is an acid labile hydroxyl protecting group; and
T3 is a base labile hydroxyl protecting group or a covalent attachment to a solid support;
(b) deblocking said acid labile hydroxyl protecting group to form a deblocked hydroxyl group;
(c) treating said deblocked hydroxyl group with a further compound having the formula: 
xe2x80x83and a condensing reagent in a solvent under conditions of time, temperature and pressure effective to form an extended compound having an added H-phosphonate internucleoside linkage;
(d) optionally treating said extended compound with a capping agent to form a capped compound;
(e) optionally treating said capped compound with a silylating, agent to give a silylated compound;
(f) optionally treating said extended compound, said capped compound, or said silylated compound with an oxidizing solution inert to phosphodiester, phosphorothioate, phosphoramidate or boranophosphate internucleoside linkages, thereby oxidizing said H-phosphonate internucleoside linkages to phosphodiester, phosphorothioate, phosphoramidate or boranophosphate internucleoside linkages;
(g) optionally repeating steps (b), (c), (d), (e) and (f) to form a protected oligomeric compound; and
(h) treating said protected oligomeric compound with a deblocking solution to form said mixed backbone oligomeric compound.
In a preferred embodiment, the mixed backbone oligomeric compound comprises contiguous regions of phosphodiester and phosphorothioate internucleoside linkages.
In another preferred embodiment, the mixed backbone oligomeric compound comprises contiguous regions of phosphodiester and phosphoramidate internucleoside linkages.
In a further preferred embodiment, the oxidizing solution comprises an oxidizing agent, an aprotic organic solvent, a base and water.
In one embodiment of the present invention the oxidizing solution comprises from about 18% to 45% oxidizing agent. In a preferred embodiment the oxidizing solution comprises from about 26% to about 40% oxidizing agent. In a more preferred embodiment the oxidizing solution comprises about 33% oxidizing agent.
The present invention also provides compounds of formula: 
wherein:
T4 is hydroxyl or a protected hydroxyl;
Bx is a heterocyclic base moiety; and
R2 has one of formula I or II: 
wherein:
E is C1-C10 alkyl, N(R4)(R5) or Nxe2x95x90C(R4)(R5);
R3 is OX, SX, or N(X)2;
each R4 and R5 is, independently, H, C1-C10 alkyl, a nitrogen protecting group, or R4 and R5, together, are a nitrogen protecting group, or R4 and R5 are joined in a ring structure that can include at least one additional heteroatom selected from a group consisting of nitrogen and oxygen;
each X is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(xe2x95x90NH)N(H)Z, C(xe2x95x90O)N(H)Z or OC(xe2x95x90O)N(H)Z;
Z is H or C1-C8 alkyl;
L1, L2 and L3 comprise a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and at least 1 heteroatom, said heteroatom being selected from a group consisting of oxygen, nitrogen and sulfur, said ring system being aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated is heterocyclic;
Y is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R4)(R5) OR4, halo, SR4 or CN;
each q1 is, independently, an integer from 2 to 10;
each q2 is, independently, 0 or 1;
p is an integer from 0 to 10; and
r is an integer from 1 to 10,
provided that when p is 0, r is an integer greater than 1.
In a preferred embodiment R2 is a 2xe2x80x2-substituent group. In another preferred embodiment the 2xe2x80x2-substituent group is xe2x80x94Oxe2x80x94CH2CH2xe2x80x94Oxe2x80x94CH3, xe2x80x94Oxe2x80x94CH2CH2xe2x80x94Oxe2x80x94N (R6) (R7) or xe2x80x94Oxe2x80x94CH2CH2xe2x80x94Oxe2x80x94CH2CH2xe2x80x94N(R6)(R7), wherein each of R6 and R7 is, independently, H or C1-C10 alkyl. In a further preferred embodiment each R6 and R7 is xe2x80x94CH3.
The present invention also provides chimeric oligomeric compounds having the formula:
5xe2x80x2xe2x80x94Nuxe2x80x94(L4xe2x80x94Nu)z1xe2x80x94(L5xe2x80x94Nu)z2xe2x80x94(L6xe2x80x94Nu)z3xe2x80x943xe2x80x2
wherein:
each L4, L5, and L6 is, independently, an internucleoside linkage selected from phosphodiester, phosphorothioate, phosphoramidate or boranophosphate, with the provision that L4 and L6 are different from L5 and that one of L4, L5 and L6 are other than phosphodiester and phosphorothioate;
each z1, z2, and z3 is, independently, an integer greater than 1;
each Nu is a nucleoside having the formula: 
xe2x80x83wherein:
Bx is a heterocyclic base moiety;
each R1 is, independently, H, hydroxyl, a protected hydroxyl, a 2xe2x80x2-substituent group or a protected 2xe2x80x2-substituent group; and
said 5xe2x80x2- and said -3xe2x80x2 ends of said chimeric oligomeric compound are, independently, hydroxyl, a protected hydroxyl, a linkage to a solid support, an activated phosphate group, an activated phosphite group, a reactive group for forming an internucleotide linkage, a nucleotide, a nucleoside, or an oligonucleotide.
In one embodiment, each L4 and L6 is a phosphoramidate internucleotide linkage and each L5 is a phosphorothioate internucleoside linkage.
In another embodiment, each L4 and L6 is a phosphoramidate internucleotide linkage and each L5 is a phosphodiester internucleoside linkage.
In yet another embodiment, each L4 and L6 is a boranophsphate internucleotide linkage and each L5 is a phosphodiester internucleoside linkage.
In a further embodiment, each L4, L5 and L6 is phosphoramidate, phosphorothioate or phosphodiester.
It is preferred that each R1 be, independently, xe2x80x94Oxe2x80x94CH2CH2xe2x80x94Oxe2x80x94CH3, xe2x80x94Oxe2x80x94CH2CH2xe2x80x94Oxe2x80x94N(R6)(R7) or xe2x80x94Oxe2x80x94CH2CH2xe2x80x94Oxe2x80x94CH2CH2xe2x80x94N(R6)(R7). It is also preferred that each of R6 and R7 be, independently, H or C1-C10 alkyl.
It is further preferred that each R6 and R7 be xe2x80x94CH3.
The present invention provides compounds and methods for the preparation of mixed backbone oligomeric, or chimeric, compounds having phosphodiester internucleoside linkages in addition to phosphorothioate and/or phosphoramidate internucleoside linkages. The methods also include incorporation of boranophosphate internucleoside linkages into oligomeric compounds of the invention. The methods utilize H-phosphonate intermediates that are coupled together forming contiguous regions of one or more H-phosphonate internucleoside linkages. Each contiguous region is subsequently oxidized to phosphodiester, phosphorothioate, phosphoramidate or boranophosphate internucleoside linkages prior to further elongation. Mixed backbone oligomeric compounds are prepared in this maimer by oxidizing adjacent regions with different reagents. Oligomeric compounds of the invention are prepared using novel oxidation steps that oxidize a region of one or more H-phosphonate internucleoside linkages without degrading existing linkages that have been previously oxidized.
The H-phosphonate method of oligomer synthesis has been used for the preparation of oligonucleotides and their analogs having a variety of internucleotide linkages. These internucleotide linkages have included phosphorothioate, phosphoramidate, methyl phosphate, methylphosphonate, alkylphosphonate, S-aryl phosphorothioate, acylphosphonate, phosphorofluoridate, phosphorodithioate, selenophosphate, (hydroxymethyl)phosphonate and boranophosphate.
As used herein, the term xe2x80x9cchimeric oligomeric compoundxe2x80x9d or xe2x80x9cmixed backbone oligomeric compoundxe2x80x9d refers to oligomeric compounds comprising nucleoside monomer subunits and containing at least two different internucleoside linkages. The mixed backbone oligomeric compounds of the present invention may contain a plurality of nucleoside monomer subunits that are joined together by more than one type of internucleoside linkages. At least one internucleoside linkage is a phosphodiester linkage and at least one other linkage is a phosphorothioate, a phosphoramidate or a boranophosphate internucleoside linkage. Thus the term xe2x80x9coligomeric compoundxe2x80x9d includes oligonucleosides, oligonucleotides, their analogs, and synthetic oligonucleotides.
In some preferred embodiments, a first monomer attached to a solid support is elongated using H-phosphonate monomers. The methods of the present invention have been used with automated DNA synthesizers to synthesize mixed backbone oligomeric compounds of desired length and sequence. Some oligomeric compounds have been synthesized having only phosphodiester and phosphorothioate internucleoside linkages. Other oligomeric compounds have been synthesized having phosphodiester, phosphorothioate and phosphoramidate internucleoside linkages. Example 14 illustrates the synthesis of a boranophosphate mixed backbone oligomeric compound. Table I illustrates selected oligomeric compounds that were synthesized using methods and intermediates of the present invention.
In preferred embodiments, the methods of the present invention are employed for use in iterative solid phase oligonucleotide synthetic regimes. Representative solid phase techniques are those typically employed for DNA and RNA synthesis utilizing standard H-phosphonate chemistry. The H-phosphonate method of oligonucleotide synthesis is disclosed in U.S. Pat. No. 5,149,798, issued Sep. 22, 1992, and U.S. Pat. No. 5,548,076, issued Aug. 20, 1996. The entire disclosure of each is incorporated herein by reference. A preferred synthetic solid phase synthesis utilizes H-phosphonates as activated phosphate compounds. In this technique, an H-phosphonate monomer is reacted with a free hydroxyl on the growing oligomer chain to produce an intermediate phosphite compound, which is subsequently oxidized to the Pv state. This technique is commonly used for the synthesis of several types of linkages including phosphodiester, phosphorothioate, and phosphoramidate linkages. Oxidation of H-phosphonate linkages to phosphodiester linkages using standard H-phosphonate chemistry results in the degradation of phosphorothioate and phosphoramidate linkages when present in a growing oligomeric compound. This degradation of adjacent linkages within an oligomeric compound has been a limitation to the use of standard H-phosphonate chemistry with mixed backbone syntheses.
Typically, the first step in such a process is attachment of a monomer or higher order subunit containing a protected 5xe2x80x2-hydroxyl to a solid support, usually through a linker, using standard methods and procedures known in the art. See, e.g., Oligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y., 1991, hereby incorporated by reference in its entirety. The support-bound monomer or higher order first synthon is then treated to remove the 5xe2x80x2-protecting group. Typically, this is accomplished by treatment with acid. The solid support bound monomer is then reacted with a second monomer or higher order synthon having a reactive group for forming an internucleoside linkage. In preferred embodiments the coupling reaction is performed under anhydrous conditions in the presence of a condensing reagent.
After the addition of each monomer or higher order synthon, a capping step is performed to cap unreacted hydroxyl groups. It is generally preferable to perform a capping step either prior to or after oxidation or sulfurization of the internucleoside linkage. Such a capping step is generally known to be beneficial by preventing shortened oligomer chains, by blocking chains that have not reacted in the coupling cycle. One representative reagent used for capping is acetic anhydride. Other suitable capping reagents and methodologies can be found in U.S. Pat. No. 4,816,571, issued, Mar. 28, 1989, hereby incorporated by reference in its entirety.
As used herein an xe2x80x9coxidizing solutionxe2x80x9d comprises a mixture of reagents effective to oxidize an H-phosphonate internucleoside linkage or silylated derivative thereof to a phosphodiester, phosphorothioate, phosphoramidate or boranophosphate internucleoside linkage. Such oxidation can be performed on a region of an oligomeric compound without causing degradation of other previously oxidized portions of the same oligomeric compound. Oxidation of each type of internucleoside linkage is illustrated in the examples below. Oxidation of the internucleoside linkage can be performed iteratively after each coupling step, or can be performed after a desired number or sequence of monomers have been coupled. This allows for the preparation of gapmer-type oligomeric compounds having one or more regions of one type of linkage with one or more other regions having another type of linkage. A representative example of a gapmer is an oligomeric compound having a phosphorothioate region at the 3xe2x80x2 and 5xe2x80x2 ends with a phosphodiester region located internally.
Treatment of the growing oligomeric compound with an acid removes the 5xe2x80x2-hydroxyl protecting group enabling another synthetic iteration. The growing oligomeric compound is extended with subsequent oxidation at selected stages with selected oxidation agents until an oligomeric compound of desired length is produced.
The completed oligomer is then cleaved from the solid support. The cleavage step can precede or follow deprotection of protected functional groups. The term xe2x80x9cdeblocking solutionxe2x80x9d as used herein is meant to encompass solutions routinely used for the deblocking of oligomeric compounds prepared either by solution or solid phase techniques. A common solution used for deblocking of oligomers synthesized on solid support is aqueous ammonia.
As used herein xe2x80x9cheterocyclic base moietyxe2x80x9d is intended to include nucleobases. Nucleobases useful in the compounds and methods described herein include but are not limited to adenine, guanine, cytosine, uridine, and thymine, as well as other non-naturally occurring and natural nucleobases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-halo, oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine. Further naturally- and non-naturally-occurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan et al.); in Sanghvi, in Antisense Research and Application, Chapter 15, S. T. Crooke and B. Lebleu, Eds., CRC Press, 1993; in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613-722 (particularly, pages 622 and 623); in the Concise Encyclopedia of Polymer Science and Engineering, J. I. Kroschwitz, Ed., John Wiley and Sons, 1990, pages 858-859; and in Cook, Anti-Cancer Drug Design, 1991, 6, 585-607, each of which is hereby incorporated by reference in its entirety. The term xe2x80x9cnucleosidic base moietyxe2x80x9d is further intended to include heterocyclic compounds that can serve as nucleosidic bases, including certain xe2x80x98universal basesxe2x80x99 that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases. Especially mentioned as a universal base is 3-nitropyrrole.
As used herein, the term xe2x80x9c2xe2x80x2-substituent groupxe2x80x9d includes 2xe2x80x2-sugar modifications. 2xe2x80x2-Sugar modifications of the present invention include fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, and polyethers of the formula (O-alkyl)m, where m is 1 to about 10. Preferred among these polyethers are linear and cyclic polyethylene glycols (PEGs), and PEG-containing groups, such as crown ethers and those which are disclosed by Ouchi et al., Drug Design and Discovery 1992, 9, 93; Ravasio et al., J. Org. Chem. 1991, 56, 4329; and Delgardo et. al., Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9, 249, each of which is hereby incorporated by reference in its entirety. Further sugar modifications are disclosed in Cook, Anti-Cancer Drug Design, 1991, 6, 585-607. Fluoro, O-alkyl, O-alkylamino, O-alkyl imidazole, O-alkylaminoalkyl, and alkyl amino substitutions are described in U.S. patent application Ser. No. 08/398,901, filed Mar. 6, 1995, entitled xe2x80x9cOligomeric Compounds having Pyrimidine Nucleotide(s) with 2xe2x80x2 and 5xe2x80x2 Substitutions,xe2x80x9d hereby incorporated by reference in its entirety.
Additional 2xe2x80x2-sugar modifications of use in the present invention include 2xe2x80x2-SR and 2xe2x80x2-NR2 groups, where each R is, independently, hydrogen, a protecting group or substituted or unsubstituted alkyl, alkenyl, or alkynyl. 2xe2x80x2-SR nucleosides are disclosed in U.S. Pat. No. 5,670,633, issued Sep. 23, 1997, hereby incorporated by reference in its entirety. Incorporation of 2xe2x80x2-SR monomer synthons is disclosed by Hamm et al., J. Org. Chem., 1997, 62, 3415-3420. 2xe2x80x2-NR2 nucleosides are disclosed by Goettingen, J. Org. Chem., 1996, 61, 6273-628 1; and Polushin et al., Tetrahedron Lett., 1996, 37, 3227-3230. Some representative 2xe2x80x2-sugar modifications amenable to the present invention include those having one of formula I or II: 
wherein:
E is C1-C10 alkyl, N(R4)(R5) or Nxe2x95x90C(R4)(R5);
each R4 and R5 is, independently, H, C1-C10 alkyl, a nitrogen protecting group, or R4 and R5, together, are a nitrogen protecting group, or R4 and R5 are joined in a ring structure that can include at least one additional heteroatom selected from a group consisting of nitrogen and oxygen;
R3 is OX, SX, or N(X)2;
each X is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(xe2x95x90NH)N(H)Z, C(xe2x95x90O)N(H)Z or OC(xe2x95x90O)N(H)Z;
Z is H or C1-C8 alkyl;
L1, L2 and L3 comprise a ring system having from about 4 to about 7 carbon atoms, or having from about 3 to about 6 carbon atoms and at least one heteroatom, said heteroatom being selected from a group consisting of oxygen, nitrogen and sulfur, said ring system being aliphatic, unsaturated aliphatic, aromatic, or saturated or unsaturated heterocyclic;
Y is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms, N(R4)(R5) OR4, halo, SR4 or CN;
each q1 is, independently, an integer from 2 to 10;
each q2 is, independently, 0 or 1;
p is an integer from 0 to 10; and
r is an integer from 1 to 10,
provided that when p is 0, r is an integer greater than 1.
Representative 2xe2x80x2-sugar substituents of formula I are disclosed in U.S. patent application Ser. No. 09/130,973, filed Aug. 7, 1998, entitled xe2x80x9cCapped 2xe2x80x2-Oxyethoxy Oligonucleotides,xe2x80x9d hereby incorporated by reference in its entirety.
Representative cyclic 2xe2x80x2-sugar substituents of formula II are disclosed in U.S. patent application Ser. No. 09/123,108, filed Jul. 27, 1998, entitled xe2x80x9cRNA Targeted 2xe2x80x2-Modified Oligonucleotides that are Conformationally Preorganized,xe2x80x9d hereby incorporated by reference in its entirety.
Sugars wherein the ring oxygen atom is replaced by other atoms are also amenable to the present invention. Representative substitutions for the ring oxygen include S, CH2, CHF, and CF2. See, e.g., Secrist et al., Abstract 21, Program and Abstracts, Tenth International Roundtable, Nucleosides, Nucleotides and their Biological Applications, Park City, Utah, Sep. 16-20, 1992, hereby incorporated by reference in its entirety.
As used herein, the term xe2x80x9calkylxe2x80x9d includes, but is not limited to, straight chain, branched chain, and alicyclic hydrocarbon groups. Alkyl groups of the present invention may be substituted. Representative alkyl substituents are disclosed in U.S. Pat. No. 5,212,295, at column 12, lines 41-50, hereby incorporated by reference in its entirety. As used herein, the term xe2x80x9clower alkylxe2x80x9d is intended to mean alkyl having 6 or fewer carbons.
As used herein, the term xe2x80x9caralkylxe2x80x9d denotes alkyl groups which bear aryl groups, for example, benzyl groups. The term xe2x80x9calkarylxe2x80x9d denotes aryl groups which bear alkyl groups, for example, methylphenyl groups. As used herein the term xe2x80x9carylxe2x80x9d denotes aromatic cyclic groups including but not limited to phenyl, naphthyl, anthracyl, phenanthryl, and pyrenyl.
As used herein, the term xe2x80x9calkanoylxe2x80x9d has its accustomed meaning as a group of formula xe2x80x94C(xe2x95x90O)-alkyl. A preferred alkanoyl group is the acetyl group.
In general, the term xe2x80x9cheteroxe2x80x9d denotes an atom other than carbon, preferably but not exclusively nitrogen, oxygen or sulfur. Accordingly, the term xe2x80x9cheterocycloalkylxe2x80x9d denotes an alkyl ring system having one or more heteroatoms, i.e., non-carbon atoms. Preferred heterocycloalkyl groups include, for example, morpholino groups. As used herein, the term xe2x80x9cheterocycloalkenylxe2x80x9d denotes a ring system having one or more double bonds and one or more heteroatoms. Preferred heterocycloalkenyl groups include, for example, pyrrolidino groups.
In a preferred embodiment of the present invention, oligomer synthesis is performed on an automated synthesizer utilizing a solid support. Solid supports are substrates which are capable of serving as the solid phase in solid phase synthetic methodologies, such as those described in U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; 5,132,418; 4,725,677 and Re. 34,069. Linkers are known in the art as short molecules which serve to connect a solid support to functional groups, e.g., hydroxyl groups, of initial synthon molecules in solid phase synthetic techniques. Suitable linkers are disclosed in, for example, Oligonucleotides And Analogues A Practical Approach, Ecstein, F., Ed., IRL Press, N.Y., 1991, Chapter 1, pages 1-23.
Solid supports according to the invention include those generally known in the art to be suitable for use in solid phase methodologies including, for example, controlled pore glass (CPG), oxalyl-controlled pore glass (see, e.g., Alul et al., Nucleic Acids Research 1991, 19, 1527, hereby incorporated by reference in its entirety), TentaGel Support; an aminopolyethyleneglycol derivatized support (see, e.g., Wright et al., Tetrahedron Letters 1993, 34, 3373, hereby incorporated by reference in its entirety) and Poros; a copolymer of polystyrene/divinylbenzene.
In some embodiments of the invention, each of T1 and T2 is, independently, a protected hydroxyl group, and T3 is a hydroxyl protecting group. A wide variety of hydroxyl protecting groups can be employed in the methods of the invention. Preferably, the protecting group is stable under either acidic or basic conditions. This enables removal of a base-stable protecting group under acidic conditions, and removal of an acid-stable protecting group under basic conditions. In general, protecting groups render chemical functionalities inert to specific reaction conditions, and can be appended to and removed from such functionalities in a molecule without substantially damaging the remainder of the molecule. Representative. hydroxyl protecting groups are disclosed by Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and also in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley and Sons, New York, 199 1, each of which is hereby incorporated by reference in its entirety. Preferred protecting groups include dimethoxytrityl (DMT), monomethoxytrityl (MMT), 9-phenylxanthen-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl (Mox).
In some preferred embodiments of the invention amino groups are appended to alkyl or other groups such as, for example, 2xe2x80x2-alkoxy groups. Such amino groups are also commonly present in naturally-occurring and non-naturally-occurring nucleobases. It is generally preferred that these amino groups be in protected form during the synthesis of oligomeric compounds of the invention. Representative amino protecting groups suitable for these purposes are discussed in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 7, 2d ed, John Wiley and Sons, New York, 1991. Generally, as used herein, the term xe2x80x9cprotected,xe2x80x9d when used in connection with a molecular moiety such as xe2x80x9cnucleobase,xe2x80x9d indicates that the molecular moiety contains one or more functionalities protected by protecting groups.
There are currently many useful condensing reagents known to the art skilled that are amenable to the H-phosphonate method of oligonucleotide synthesis. Wada et al., J. Am. Chem. Soc., 1997, 119, 12710-12721. Useful condensing reagents include, but are not limited to, acid chlorides, chlorophosphates, carbonates, carbonium type compounds and phosphonium type compounds. In a preferred embodiment the condensing reagent is selected from a group consisting of pivaloyl chloride, adamantyl chloride, 2,4,6-triisopropyl-benzenesulfonyl chloride, 2-chloro-5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinane, diphenyl phosphorochloridate, bis(2-oxo-3-oxazolidinyl)phosphinic chloride, bis(pentafluorophenyl)carbonate, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, O-(azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate, 6-(trifluoromethyl)benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, bromo-tris-pyrrolidino-phosphonium hexafluorophosphate, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate and 2-(benzotriazol-1-yloxy)-1,3-dimethyl-2-pyrrolidin-1-yl-1,3,2-diazaphospholidinium hexafluorophosphate.
Sulfurizing agents used during oxidation to form phosphorothioate and phosphorodithioate linkages, when they are the only linkages present, include Beaucage reagent (Iyer et.al., J. Chem. Soc., 1990, 112, 1253-1254; and Iyer et.al., J. Org. Chem., 1990, 55, 4693-4699); tetraethylthiuram disulfide (Vu and Hirschbein, Tetrahedron Lett., 1991, 32, 3005-3008); dibenzoyl tetrasulfide (Rao, et.al., Tetrahedron Lett., 1992, 33, 4839-4842); di(phenylacetyl)disulfide (Kamer, Tetrahedron Lett., 1989, 30, 6757-6760); Bis(O,O-diisopropoxy phosphinothioyl)disulfides (Stec et al., Tetrahedron Lett., 1993, 34, 5317-5320); 3-ethoxy-1,2,4-dithiazoline-5-one (Nucleic Acids Research, 1996 24, 1602-1607; and Nucleic Acids Research, 1996 24, 3643-3644); bis(p-chlorobenzenesulfonyl)-disulfide (Nucleic Acids Research, 1995 23, 4029-4033); sulfur, and sulfur in combination with ligands such as triaryl, trialkyl, triaralkyl, or trialkaryl phosphines. Each of the foregoing references is hereby incorporated by reference in its entirety.
Useful oxidizing agents used to form the phosphodiester or phosphorothioate linkages, when they are the only linkages present, include iodine/tetrahydrofuran/water/pyridine, hydrogen peroxide/water, tert-butyl hydro peroxide or any peracid like m-chloroperbenzoic acid. In the case of oxidation using a sulfur species, the reaction is performed under anhydrous conditions with the exclusion of air, in particular oxygen, whereas in the case of oxidation the reaction can be performed under aqueous conditions.
The oxidation of H-phosphonate internucleoside linkages, without simultaneous degradation of existing, previously oxidized linkages, is performed using a novel oxidizing solution. The oxidizing solution comprises particular compounds/reagents dependent upon what oxidized linkages are being prepared from the H-phosphonate linkages. In general, the oxidation solution comprises an oxidizing agent, an aprotic organic solvent, and a base.
Bases are present in the oxidizing solution to assist in the oxidation process. Selected bases have a pka ranging from about 9 to about 12, which is believed to be the optimum range for deprotonation of H-phosphonate or water without causing cleavage of the backbone of the oligomeric compound. While not wanting to be bound by theory, it is believed that bases within this pKa range, and present in the oxidation solution in a concentration of from about 0.01 to about 0.8 M, are necessary for oxidation of H-phosphonate linkages without simultaneous degradation of existing oxidized linkages. A more preferred range for the concentration of the base is from about 0.05 to about 0.2 M in the oxidation solution. Representative bases that are useful in the present invention include 4-(dimethylamino)pyridine (DMAP), triethylamine (TEA) and N,N-diisopropylethylamine (DIEA).
Many aprotic organic solvents known to those skilled in the art are amenable to the present invention. Some representative aprotic organic solvents include pyridine, acetonitrile and dimethylformamide.
The choice of oxidizing agent is dependent on the particular internucleoside linkage that is desired for the region of H-phosphonate internucleoside linkages that is being oxidized. Representative oxidizing agents useful for the preparation of phosphodiester internucleoside linkages include carbon tetrachloride, carbon tetrabromide, N-chlorosuccinimide and N-bromosuccinimide. In addition to an oxidizing agent, water is a necessary component of the oxidizing solution for preparing phosphodiester linkages.
Phosphorothioate internucleoside linkages are prepared using an oxidizing agent that oxidizes phosphorus by addition of sulfur rather than oxygen. Elemental sulfur has been used to sulfurize H-phosphonate to form phosphorothioate internucleoside linkages as illustrated in the examples below. The use of elemental sulfur in the oxidizing solution affords the desired phosphorothioate internucleoside linkages while leaving unaffected any preexisting oxidized linkages in the same oligomeric compound.
The selective oxidation of H-phosphonate internucleoside linkages to phosphodiester, phosphorothioate, phosphoramidate and boranophosphate internucleoside linkages in a mixed backbone oligomeric compound is dependent on the oxidizing solution. The oxidizing solution must not affect preexisting linkages in the oligomeric compound that is undergoing oxidation. The oxidizing solution is a mixture of an oxidizing agent, an aprotic solvent and a base. For phosphodiester linkages water is also a component of the oxidizing solution. The concentration of reagents in the oxidizing solution for a given oxidation can vary. Optimal concentration of reagents can be discerned by the art-skilled without undue experimentation.
The composition and concentration of reagents in the oxidizing solution used to oxidize an H-phosphonate linkages in a mixed backbone oligomeric compound is variable dependent on whether the desired oxidized linkage is phosphodiester, phosphorothioate or phosphoramidate. In the case of boranophosphate internucleoside linkages, a silylation step is also involved. The two main components are the base and the oxidizing agent. For phosphodiester and phosphorothioate internucleoside linkages, an aprotic organic solvent is also used. Water is also used in the oxidizing solution when phosphodiester linkages are prepared.
The oxidizing solution used for preparing phosphodiester internucleoside linkages in a mixed backbone oligomeric compound of the invention generally has from about 18% to about 45% oxidizing agent, from about 2% to about 15% water, from about 40% to about 80% aprotic organic solvent, and from about 0.01 M to about 0.8 M base dissolved in the aprotic organic solvent. A more preferred range of concentrations is from about 26% to about 40% oxidizing agent, from about 4% to about 10% water, from about 50% to about 70% aprotic organic solvent, and from about 0.04 M to about 0.4 M base dissolved in the aprotic organic solvent. Even more preferred is about 33% oxidizing agent, about 7% water, about 60% aprotic organic solvent, and from about 0.05 M to about 0.2 M base dissolved in the aprotic organic solvent.
The oxidizing solution used for preparing phosphorothioate internucleoside linkages in a mixed backbone oligomeric compound of the invention generally has from about 1% to about 15% oxidizing agent, about 40% to about 60% solvent that will solubilize the oxidizing agent, from about 40% to about 60% aprotic organic solvent, and from about 0.01 M to about 0.8 M base dissolved in the aprotic organic solvent. A more preferred range of concentrations is from about 1% to about 10% oxidizing agent, about 40% to about 60% solvent that will solubilize the oxidizing agent, from about 40% to about 60% aprotic organic solvent, and from about 0.02 M to about 0.5 M base dissolved in the aprotic organic solvent. Even more preferred is about 3% to about 8% oxidizing agent, about 40% to about 60% solvent that will solubilize the oxidizing agent, from about 40% to about 60% aprotic organic solvent, and from about 0.04 M to about 0.1 M base dissolved in the aprotic organic solvent.
The oxidizing solution used for preparing phosphoramidate internucleoside linkages in a mixed backbone oligomeric compound of the invention generally has a primary or secondary amine in combination with an oxidizing agent. An oxidizing agent that acts as a solvent for the primary or secondary amine is preferable. A preferred oxidizing agent is carbon tetrachloride. The oxidizing solution has from about 1% to about 15% of the primary or secondary amine, and from about 85% to about 99% oxidizing agent. A preferred concentration is about 1% to about 10% of the primary or secondary amine, and from about 90% to about 99% oxidizing agent. A more preferred concentration of primary or secondary amine is about 2% to about 5% by volume in the oxidizing solution.
The conversion of H-phosphonate internucleoside linkages to the boranophosphate (Oxe2x95x90Pxe2x80x94BH3) internucleoside linkages requires an additional silylation step prior to the oxidation step. Sergueev et al., J. Am. Chem. Soc., 1998, 120, 9417-9427. A number of silylating agents have been shown to be effective in the silylation step including chlorotrimethyl-silane, N,O-bis-(trimethylsilyl)acetamide, heptamethyldisilazane and N,O-bis-(trimethylsilyl)trifluoroacetamide. The silylation step is followed by oxidation with borane-N,N-diisopropylethylamine (BH3xc3x97DIEA, 0.1 mmol to 1 mmol), borane-2-chloro-pyridine or borane-aniline.
In one aspect of the present invention, the compounds of the invention are used to modulate RNA or DNA encoding a protein whose formation or activity is to be modulated. The targeting portion of the composition to be employed is, thus, selected to be complementary to the preselected portion of DNA or RNA. That is, the targeting portion of the composition is hybridizable to the preselected portion of DNA.
The methods of the present invention can be used to prepare oligomeric compounds that are used in diagnostics, therapeutics, and as research reagents and kits. They can be used in pharmaceutical compositions by including a suitable pharmaceutically acceptable diluent or carrier. They further can be used for treating organisms having a disease characterized by the undesired production of a protein. The organism should be contacted with an oligonucleotide having a sequence that is capable of specifically hybridizing with a strand of nucleic acid encoding the undesirable protein. Treatments of this type can be practiced on a variety of organisms ranging from unicellular prokaryotic and eukaryotic organisms to multicellular eukaryotic organisms. Any organism that utilizes DNA-RNA transcription or RNA-protein translation as a fundamental part of its hereditary, metabolic or cellular control is susceptible to therapeutic and/or prophylactic treatment in accordance with the invention. Seemingly diverse organisms such as bacteria, yeast, protozoa, algae, all plants and all higher animal forms, including warm-blooded animals, can be treated. Further, each cell of multicellular eukaryotes can be treated, as they include both DNA-RNA transcription and RNA-protein translation as integral parts of their cellular activity. Furthermore, many of the organelles (e.g., mitochondria and chloroplasts) of eukaryotic cells also include transcription and translation mechanisms. Thus, single cells, cellular populations or organelles can also be included within the definition of organisms that can be treated with therapeutic or diagnostic oligonucleotides.